The present invention relates to a method of treating or preventing a disease state associated with a genitourinary or pain disorder using a purinergic receptor modulator. Also provided are transgenic or knockout animals deficient in a purinergic receptor and a method of screening for therapeutic compounds using the transgenic or knockout animals as positive controls.
The urinary bladder is responsible for two important physiological functions: urine storage and urine emptying. This process involves two main steps: (1) the bladder fills progressively until the tension in its walls rises above a threshold level; and (2) a nervous reflex, called the micturition reflex, occurs that empties the bladder or, if this fails, at least causes a conscious desire to urinate. Although the micturition reflex is an autonomic spinal cord reflex, it can also be inhibited or mediated by centers in the cerebral cortex or brain.
Purines, acting via extracellular purinoreceptors, have been implicated as having a variety of physiological and pathological roles. (See, Burnstock (1993) Drug Dev. Res. 28:195-206.) ATP, and to a lesser extent, adenosine, can stimulate sensory nerve endings resulting in intense pain and a pronounced increase in sensory nerve discharge. ATP receptors have been classified into two major families, the P2Y- and P2X-purinoreceptors, on the basis of molecular structure, transduction mechanisms, and pharmacological characterization. The P2Y-purinoreceptors are G-protein coupled receptors, while the P2X-purinoreceptors are a family of ATP-gated cation channels. To date, cDNAs for seven P2X receptors subtypes have been cloned: P2X1; P2X2; P2X3; P2X4; P2X5; P2X6; and P2X7. (See, Chen, et al. (1995) Nature 377:428-431; Lewis, et al. (1995) Nature 377:432-435; and Burnstock (1997) Neurophamacol. 36:1127-1139.) The structure and chromosomal mapping of mouse genomic P2X3 receptor subunit has also been described. (See, Souslova, et al. (1997) Gene 195:101-111.) Purinergic receptors, in particular, P2X receptors, are known to form homomultimers or heteromultimers. In vitro, co-expression of P2X2 and P2X3 receptor subunits is necessary to produce ATP-gated currents with the properties seen in some sensory neurons. (See, Lewis, et al. (1995) Nature 377:432-435.)
Data exists suggesting that ATP may be released from epithelial/endothelial cells of various hollow organs, e.g., the urinary bladder, as a result of distention. (See, Burnstock (1999) J. Anatomy 194:335-342; and Ferguson et al. (1997) J. Physiol. 505:503-511.) Further studies propose that ATP released in this manner could serve a role in conveying information to sensory neurons located in subepithelial components, e.g., suburothelial lamina propria. (See Namasivayam, et al. (1999) BJU Intl. 84:854-860.) The P2X receptors have been studied in a number of neurons, including sensory, sympathetic, parasympathetic, mesenteric, and central neurons. (See, Zhong, et al. (1998) Br. J. Pharmacol. 125:771-781.) Recent evidence also suggests a role of endogenous ATP and purinergic receptors in nociceptive responses in mice. (See, Tsuda, et al. (1999) Br. J. Pharmacol. 128:1497-1504.)
However, while these studies suggest that purinergic receptors may play a role in afferent neurotransmission from the bladder, the exact molecular mechanism, i.e., the particular P2X receptor subunit or the involvement of this receptor subtype in the afferent control of the bladder, has yet to be defined.
The present invention fills this need by providing evidence that P2X receptor complexes, where the receptor complex comprises at lease one P2X3 receptor subunit, play a critical role in relaying bladder filling information to the central nervous system. Therefore modulation of receptor complexes comprising at least one P2X3 receptor subunit, either through antagonist- or agonist-mediated desensitization, may have utility in treating disease states associated with genitourinary disorders. Disease states associated with pain, in particular peripheral pain, inflammatory pain, or tissue injury pain can also be treated using a P2X3 receptor subunit modulator.
The present invention provides a method of treating a subject having a disease state associated with a genitourinary or pain disorder comprising administering to the animal an effective amount of a purinoreceptor modulator. In further embodiments the genitourinary disorder is an overactive bladder, outlet obstruction, outlet insufficiency, or pelvic hypersensitivity. In a further embodiment, the pain disorder is peripheral pain, inflammatory pain, or tissue injury pain. In another embodiment the purinoreceptor modulator is a P2X receptor complex modulator. Preferably, the P2X receptor complex comprises at least one P2X3 subunit. The present invention also provides that the P2X receptor complex modulator is an antagonist. In another embodiment the P2X receptor complex modulator is an agonist. In a further embodiment, the subject is a mammal, preferably a human.
The present invention encompasses a transgenic animal containing an altered allele for the gene that naturally encodes and expresses a functional P2X3 purinoreceptor subunit. In another embodiment, the altered allele is a non-functional allele. In a further embodiment, the transgenic animal is a knockout (KO) animal. The KO animal has a phenotype, when compared to a wild-type control animal, which comprises: an increase in urinary bladder capacity; a lower frequency of urine voiding; larger voided volumes; and no significant change in cystometric pressure. In another embodiment, the KO animal has a phenotype when compared to a wild-type animal comprising attenuated nociception in response to ATP injection or attenuated nociception in response to formalin injection. In a further embodiment, KO transgenic animal is a mouse.
The present invention provides a method for selecting a potential therapeutic compound for use in the treatment of a disease state associated with a genitourinary disorder comprising: administering the compound to a wild-type animal or an animal having a disease state associated with a genitourinary disorder; measuring the resulting phenotype of the wild-type animal or the animal having the disease state; and comparing the resulting phenotype of the wild-type animal or the animal having the disease state to the phenotype of the KO animal.
The present invention further provides a method for selecting a potential therapeutic compound for use in the treatment of a disease state associated with a pain disorder comprising: administering the compound to a wild-type animal or an animal having a disease state associated with a pain disorder; measuring the resulting phenotype of the wild-type animal or the animal having the disease state; and comparing the resulting phenotype of the wild-type animal or the animal having the disease state to the phenotype of the KO animal.